The present invention relates to novel enone reductases which are useful for reducing an xcex1,xcex2-unsaturated bond of an xcex1,xcex2-unsaturated ketone (enone), and polynucleotides encoding such reductases, as well as methods for producing the reductases and methods for selectively reducing a carbon-carbon double bond of an xcex1,xcex2-unsaturated ketone using the reductases or a polypeptide having homology with the enzyme.
Ketones are compounds that are widely used as raw materials in the synthesis of organic compounds. In addition, ketones are also important raw materials for the production of optically active alcohols and optically active amines that are optically active intermediates important in the synthesis of pharmaceuticals. For example, xcex1,xcex2-unsaturated ketones obtainable by the condensation reaction of aldehydes and ketones are useful as precursors for these ketones.
For example, 3-methyl-3-penten-2-one can be readily prepared by the condensation of acetaldehyde and 2-butane (J. Amer. Chem. Soc., 81:1117-1119, 1959).
Various ketones can be obtained by selectively reducing the xcex1,xcex2-unsaturated bonds of xcex1,xcex2-unsaturated carbonyl compounds. Hydrogenation reactions using Ni catalyst or Pd-C catalyst (xe2x80x9cCatalytic Hydrogenation Reactionxe2x80x9d p135, Tokyo Kagaku Dojin (1987)) are methods known in the art for selectively reducing the xcex1,xcex2-unsaturated bonds alone, without reducing any carbonyl groups. However, these methods have the following problems to be solved: (1) carbonyl groups may be also reduced by continuing the reaction; (2) metals, which have adverse effects on the environment, are used as the catalysts; and (3) high-pressure hydrogen gas is required for the reaction. Importantly, the reduction of carbonyl groups leads to decrease of the ketone yield.
On the other hand, methods using organisms as follows are reported as methods for selectively reducing carbon-carbon double bonds of xcex1,xcex2unsaturated ketones using biological reactions:
plant cells (J. Nat. Prod. 56:1406-1409, 1993);
baker""s yeast (Tetrahedron Lett. 52:5197-5200, 1978; Bull. Chem. Soc. Jpn. 64:3473-3475, 1991; Tetrahedron Asym. 6:2143-2144, 1995; etc.); and
fungus (J. Org. Chem. 47:792-798, 1982).
However, these biological methods have their own problems such as: (1) carbonyl groups are also reduced; (2) low reactivity; and (3) cell preparation on a large-scale is difficult. Further, various types of enone reductases derived from these organisms have been reported. However, genes encoding these reductases remain to be cloned, and it is therefore hard to conveniently prepare these enzymes on a large scale.
In addition to the above-mentioned reductases of the xcex1,xcex2-unsaturated carbonyl compounds, such reductases as follows have been reported. These reductases are not suitable for industrial applications because either the substrate specificity of these reductases remains to be clarified or the selectivity for the xcex1,xcex2-unsaturated bond is low.
Clostridium tyrobutyricum-derived 2-enoate reductase (E.C.1.3.1.31) (J. Biotechnol. 6:13-29, 1987);
Clostridium kluyveri-derived acryloyl-CoA reductase (Biol. Chem. Hoppe-Seyler 366:953-961, 1985);
Enone reductase YER-2 purified from baker""s yeast (Kawai et al. ((Kyoto University), The 4th Biocatalyst symposium, Abstract p58 (2001));
Enone reductases purified from a baker""s yeast EI and EII (Eur. J. Biochem. 255:271-278, 1998);
Enone reductase (verbenone reductase; also referred to as p90) derived from tobacco (Nicotiana tabacum) cells (J. Chem. Soc., Chem. Commun. 1426-1427, 1993; Chem. Lett. 850-851, 2000);
Carvone reductase (also referred to as enone reductase-I), which is an enone reductase derived from tobacco (Nicotiana tabacum) cells (Phytochemistry 31:2599-2603, 1992):
Enone reductase-II, p44, and p74, which are enone reductases derived from tobacco (Nicotiana tabacum) cells;
Enone reductases purified from Euglena gracilis and Astasia longa, which are plant species (Phytochemistry 49, 49-53 (1998)); and
Enone reductase purified from rat liver (Arch. Biochem. Biophys. 282:183-187, 1990).
The object of the present invention is to provide novel enone reductases, which have an enzyme activity to selectively reduce the xcex1,xcex2-unsaturated bonds of xcex1,xcex2-unsaturated ketones to produce xcex1,xcex2-saturated ketones, and genes encoding the reductases. Another object of the present invention is to provide methods for selectively reducing the carbon-carbon double bonds of xcex1,xcex2-unsaturated ketones using the reductases and organisms producing the reductases.
The present inventors screened enzymes producing 2-butanone from methyl vinyl ketone and found that Kluyveromyces lactis has the activity of interest. Then, they purified the enzyme having the activity of interest from fungal cells of Kluyveromyces lactis, and revealed the properties thereof. They confirmed that the enzyme selectively reduced the xcex1,xcex2-unsaturated bonds of xcex1,xcex2-unsaturated ketones in a xcex2-nicotinamide adenine dinucleotide phosphate (NADPH)-dependent manner, and that the enzyme has substantially no activity to reduce ketones. Further, the present inventors cloned a gene encoding the enzyme, clarified the structure thereof, and verified that the gene was novel. In addition, they overexpressed the gene in a heterologous organism to obtain a transformed strain having higher selectivity and higher activity at the same time to reduce the xcex1,xcex2-unsaturated bonds of xcex1,xcex2-unsaturated ketones in a NADPH-dependent manner. Furthermore, they found that selective reduction of the carbon-carbon double bonds of xcex1,xcex2-unsaturated ketones can be achieved by the enzyme, homologues thereof, cells producing them, and so on, and thus, completed the present invention. Hereinafter, xcex2-nicotinamide adenine dinucleotide phosphate is referred to as NADP; xcex2-nicotinamide adenine dinucleotide as NAD; and the reduced forms thereof as NADPH and NADH, respectively.
More specifically, the present invention relates to the following enone reductases, polynucleotides encoding the reductases, methods for producing the reductases, and methods for selectively reducing carbon-carbon double bonds of xcex1,xcex2-unsaturated ketones using the reductases or polypeptides having homology to such reductases.
[1] An enone reductase having the following physicochemical properties:
(A) Action:
The enzyme reduces the carbon-carbon double bonds of the xcex1,xcex2-unsaturated ketones, using NADPH as an electron donor, to produce the corresponding saturated hydrocarbon;
(B) Substrate specificity:
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
(1) the enzyme reduces the carbon-carbon double bonds of the xcex1,xcex2-unsaturated ketones but has substantially no activity to reduce ketones;
(2) the enzyme exhibits a significantly higher activity with NADPH than with NADH as the electron donor;
(3) the enzyme does not substantially act on substrates, wherein both substituents at the xcex2 carbon relative to the ketone are not hydrogen; and
(4) the enzyme does not substantially act on substrates, wherein the carbon-carbon double bond is present in the cyclic structure; and
(C) Optimal pH:
pH 6.5-7.0;
[2] The enone reductase of [1], wherein the reductase further has the following physicochemical properties:
(D) Optimum temperature:
37-45xc2x0 C.
(E) Molecular weight:
The molecular weight of the reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration is about 43,000 and about 42,000, respectively;
[3] The novel enone reductase of [1], which is derived from the genus Kluyveromyces;
[4] A method for obtaining the enone reductase of [1], comprising the step of culturing a microorganism belonging to the genus Kluyveromyces and having the ability of producing to the novel enone reductase of [1];
[5] The method of [4], wherein the microorganism belonging to the genus Kluyveromyces is Kluyveromyces lactis; 
[6] A polynucleotide encoding a polypeptide having enone-reducing activity selected from the group of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1;
(b) a polynucleotide encoding the amino acid sequence of SEQ ID NO:2;
(c) a polynucleotide encoding a polypeptide comprising the the amino acid sequence of SEQ ID NO:2, in which one or more amino acids are substituted, deleted, inserted, and/or added;
(d) a polynucleotide hybridizing under stringent conditions with a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:1; and
(e) a polynucleotide encoding an amino acid sequence exhibiting 60% or higher percent identity to the amino acid sequence of SEQ ID NO:2;
[7] A polypeptide encoded by the polynucleotide of [6];
[8] A recombinant vector comprising the polynucleotide of [6];
[9] The recombinant vector of [8], wherein a polynucleotide encoding a dehydrogenase catalyzing oxidation-reduction reactions using NADP as a coenzyme is further inserted;
[10] A transformant harboring the polynucleotide of [6] or the vector of [8] in an expressible manner;
[11] A method for producing the polypeptide of [7], comprising the step of culturing the transformant of [10];
[12] A polynucleotide encoding a polypeptide having enone-reducing activity selected from the group of:
(a) a polynucleotide comprising the nucleotide sequence of any one of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7;
(b) a polynucleotide encoding a polypeptide comprising the amino acid sequence of any one of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.
(c) a polynucleotide encoding the amino acid sequence comprising the sequence of any one of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, in which one or more amino acids are substituted, deleted, inserted and/or added;
(d) a polynucleotide hybridizing under stringent conditions with a polynucleotide consisting of the nucleotide sequence of any one of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7; and
(e) a polynucleotide encoding an amino acid sequence exhibiting 60% or higher percent identity to the amino acid sequence of any one of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8;
[13] A polypeptide encoded by the polynucleotide of [12];
[14] A recombinant vector wherein the polynucleotide of [12] has been inserted;
[15] The recombinant vector of [14], wherein a polynucleotide encoding a dehydrogenase catalyzing oxidation-reduction reactions using NADP as a coenzyme is further inserted;
[16] A transformant harboring the polynucleotide of [12] or the vector of [14] in an expressible manner;
[17] A method for producing the polypeptide of [13], comprising the step of culturing the transformant of [16];
[18] A method for selectively reducing the carbon-carbon double bonds of xcex1,xcex2-unsaturated ketones comprising the step of reacting the xcex1,xcex2-unsaturated ketones with enzyme active materials selected from the group of: (1) enone reductase of [1]; (2) the polypeptide of [7]; (3) the polypeptide of [13]; (4) a microorganism producing the enzyme or polypeptide; and (5) processed products of the microorganism; and
[19] The method of [18], wherein the microorganism producing the enzyme or polypeptide is the transformant of [10] and/or [16].